Producing DNA for metagenomic sequencing

The goal is to generate short-read metagenomes from intact thalli. The metagenome-assembled genomes (MAGs) from these can be used to improve the analysis of existing RNA-seq data from lichen thalli. Plan to use 9 thalli in total (6 freshly collected, 3 from the growth chamber)

Collecting samples

Fresh samples

First batch

  • Collected on 4.11.2022 at the TSL campus
  • Six samples in total, three from concrete (GT0240-242) & three from tree bark (GT0243-245)

Second batch

  • Collected on 17.11.2022
  • Same location
  • Two samples: GT0249 from tree bark (same tree as GT0245), GT0250 from concrete (same fence post as GT0241)

drawing

drawing

Samples from growth chambers

  • Picked 3 samples from the growth chamber (Ground floor TSL, TSL05)
    • growth chamber 1: from the petri dish labelled “Xp + Tr1”.
    • drawing
      • growth chamber 2: from L17 branch (the thallus collected from the opposite side of the branch from the label)
      • growth chamber 3: from L22 branch
  • All collected by Klara on the TSL campus

Info on used samples

See ../analysis_and_temp_files/01_DNA_extraction/metagenomes_sample_info.txt

specimen_id species substrate sample_description collection_date location
GT0240 Xanthoria parietina concrete concrete_1 04/11/2022 TSL campus, near the entrance from Colney lane, gate on the road leading to Hill house, concrete fence post on the north side of the gate
GT0241 Xanthoria parietina concrete concrete_2 04/11/2022 TSL campus, near the entrance from Colney lane, fence connected to the gate on the road leading to Hill house, third concrete fence post to the south from the gate
GT0242 Xanthoria parietina concrete concrete_3 04/11/2022 TSL campus, near the entrance from Colney lane, fence connected to the gate on the road leading to Hill house, corner fence post
GT0243 Xanthoria parietina tree bark treebark_1 04/11/2022 TSL campus, near the entrance from Colney lane, first oak to the south from the road leading to Hill house
GT0244 Xanthoria parietina tree bark treebark_2 04/11/2022 TSL campus, near the entrance from Colney lane, third oak to the south from the road leading to Hill house
GT0245 Xanthoria parietina tree bark treebark_3 04/11/2022 TSL campus, near the entrance from Colney lane, young oak between John Innes Centre and the parking lot
GT0246 Xanthoria parietina tree bark growthchamber_1 04/11/2022 Collected at the TSL campus, were stored in a growth chamber (ground floor double-door incubator in the growth chamber room)
GT0247 Xanthoria parietina tree bark growthchamber_2 04/11/2022 Collected at the TSL campus, were stored in a growth chamber (ground floor double-door incubator in the growth chamber room)
GT0248 Xanthoria parietina tree bark growthchamber_3 04/11/2022 Collected at the TSL campus, were stored in a growth chamber (ground floor double-door incubator in the growth chamber room)
GT0249 Xanthoria parietina tree bark treebark_4 17/11/2022 TSL campus, near the entrance from Colney lane, young oak between John Innes Centre and the parking lot (same as treebark_3)
GT0250 Xanthoria parietina concrete concrete_4 17/11/2022 TSL campus, near the entrance from Colney lane, fence connected to the gate on the road leading to Hill house, third concrete fence post to the south from the gate (same as concrete_2)
GT0251 Xanthoria parietina tree bark growthchamber_4 27/01/2023 Collected at the TSL campus, were stored in a growth chamber (ground floor double-door incubator in the growth chamber room)
GT0252 Xanthoria parietina tree bark growthchamber_5 27/01/2023 Collected at the TSL campus, were stored in a growth chamber (ground floor double-door incubator in the growth chamber room)

Extracting DNA

Lab notes, chronologically organized

08-09.11.2022

  • Snapfrozen the samples in liquid nitrogen and pulverised them using Geno/grinder for 1 min on 1,200 rpm
  • Used Qiagen DNEasy plant mini kit with extended incubation time (~ 1h)
  • QC’d the DNA extractions using Nanodrop and Qubit (see ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.08.csv)

Conclusion: DNA is mostly good, but the concentration is below the 10 ng/ul requerement. Will speedvac and qubit again

11.11.2022

  • Speedvaced all extractions but 2 (GTX0468 and GTX0470 had >10 ng/ul)
  • New Nanodrop and Qubit results (see ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.11.csv)
    • two extractions were still on the lower concentration threshold. three more don’t have good purity scores
  • Re-prepped extractions that have low concentration and/or bad Nanodrop scores:
    1. concrete2 (GT0241)
    2. growth chamber 1 (different thallus from GTX0471! can’t be pooled!)
    3. treebark3 (GT0245)
    4. growth chamber 2 (same thallus from GTX0472)
    5. growth chamber 3 (same thallus from GTX0473)
  • Re-extracted using same protocol, except with the addition of RNAse
  • All failed (weird curves)

14.11.2022

  • Tried diluting the three extractions speedvaced on 11.11 (GTX0467, GTX0472, GTX0473). Goal: normalize the purity scores (which likely worsened due to speedvac)

  • QCd them on nanodrop and qubit (see ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.14.csv)

  • Conclusion: QC scores improved, but concentrations are too low now

  • Re-prepped the same 5 extractions:

    1. concrete2 (GT0241)
    2. treebark3 (GT0245)
    3. growth chamber 1 (GT0246; different thallus from GTX0471! can’t be pooled!)
    4. growth chamber 2 (GT0247; same thallus from GTX0472)
    5. growth chamber 3 (GT0248; same thallus from GTX0473)

  • Extracted them using the same kit, this time with no RNAse. Diluted in 50 uL of TE

  • QCd the extractions. Nanodrop and Qubit results (see ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.14.csv)

    • looks good, but concentrations are too low

15.11.2022

  • Speevaced 8 samples:
    • Three from 09.11 (GTX0467, GTX0472, GTX0473). They have been speedvaced and diluted before
    • Five from 14.11 (GTX0479-GTX0483). These are all new extractions produced on that day
  • QCd them again. See the results in ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.15.csv

16.11.2022

  • Pooled together DNA extractions for:
    • GT0247 (growthchamber 2): GTX0472 and GTX0482
    • GT0248 (growthchamber 3): GTX0473 and GTX0483
  • Speedvaced them
  • QCd them again. See the results in ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.16.csv

18.11.2022

  • Extracted DNA from the second batch samples
    • GT0249 (treebark_4)
      • GTX0484 (eluted DNA in 50 uL of TE buffer)
      • GTX0485 (eluted DNA in 50 uL of nuclease-free water)
    • GT0250 (treebark_4)
      • GTX0486 (eluted DNA in 50 uL of TE buffer)
      • GTX0487 (eluted DNA in 50 uL of nuclease-free water)
  • Ran Nanodrop on all 4 extraction. See the results in ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.18.csv
  • Pooled GTX0486 and GTX0487 (both from GT250, one was diluted in TE and the other in water) and speedvaced
  • QCd the extractions, see in ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2022.11.18.csv

17.01.2023

  • Replaced GTX0470 (concrete 3) with diluted GTX0491
  • GTX0491 was extracted from GT0242 (concrete 3) using Nuclobond HMW kit, for the Nanopore sequencnig
  • To be on the safe side, decided to use the same extraction for Illumina sequencing
  • Diluted it 3 times (10 uL of extraction + 20 uL of HE buffer), to the final concentration 36 ng/uL

QC

  • Ran a gel to check DNA quality
  • Used two extractions: GTX0466 (fresh Xanthoria thallus) and GTX0472-482 (from growth chamber)
  • GTX0466 looks good, single band around 20 Kbp, little smear
  • GTX0472-482 looks worse, no visible band
  • Will send all anyways, and see what Novogene’s QC results will be like

Round-up of DNA for the metagenomic survey

See ../analysis_and_temp_files/01_DNA_extraction/metagenomic_dna_roundup.csv

Extraction.Name Sample.ID Sample.description Qubit.concentration..ng.uL. A260.A280 A260.A230 Avg.fragment.length..bp. Volume.shipped..uL.
GTX0465 GT0243 tree bark 1 39.8 2.06 1.97 NA 30
GTX0466 GT0244 tree bark 2 36.9 2.10 1.92 NA 30
GTX0484 GT0249 tree bark 4 20.0 2.14 2.53 NA 30
GTX0468 GT0240 concrete 1 19.8 2.08 2.13 NA 30
GTX0486, GTX0487 GT0250 concrete 4 21.1 1.98 2.22 NA 30
GTX0491_diluted GT0242 concrete 3 36.0 1.87 2.02 NA 30
GTX0481 GT0246 growth chamber 1 14.4 2.01 1.75 NA 25
GTX0472, GTX0482 GT0247 growth chamber 2 16.0 2.02 1.42 NA 25
GTX0473, GTX0483 GT0248 growth chamber 3 18.3 2.06 1.52 NA 25

NB! In addition to these 9 metagenomes, in the same run I sent genomic DNA from Trebouxia pure culture (GTX0488). This extraction had similar concentration to GTX0491 and was treated the same (including the dilution step). See ../../02_long_read_assemblies/analysis_and_temp_files/01_DNA_extraction for the details

Replacing bad-quality samples

  • 27.01.2023
  • After Novogene ran sample QC, they identified 2 samples that didn’t pass the check. Both came from the grow-chamber lichen material
    • pooled GTX0472+482
    • pooled GTX0473+483
  • Extracted DNA from 2 other lichen thalli from the growth chambers (GT0251-252)
  • See ../analysis_and_temp_files/01_DNA_extraction/nanodrop_qubit_2023.01.27.csv for all DNA extractions info and analysis_and_temp_files/01_DNA_extraction/SIFTNA* for the info submitted to Novogene
  • Send to Novogene two samples: GTX0493 (extracted from GT0251, speedvaced, new concentration should be about 14 ng/uL) and GTX494 (extracted from GT0252, concentration 20.7 ng/uL)